Figure 2
Efficient bi-allelic targeting in human iPSC and problematic FIAU-based counter-selection.
(A) Schematic of correctly integrated AAT-PB-PGK-puroΔtk donor in the human SERPINA1 locus and of untargeted allele. (B) PCR analysis of puromycin selected human iPSC clones from a patient with severe ZZ-A1AT deficiency (hPi) transfected with and Cas9 nuclease + AAT_g1 or Cas9_D10A + AAT_g1 or Cas9_D10A + AAT_g1 + AAT_g2 gRNAs. Primers for detection of integrated flanking arms are matching the genomic regions up- and downstream of the flanking arms, respectively, and inside the selection cassette. Primers for detection of untargeted allele (uta) generate a short fragment in the genomic region corresponding to the left and right flanking arms. Mono-allelic targeted clones show all three bands: left, right and uta (framed in pink), whereas bi-allelic targeted clones show absence of the uta band (framed in blue). Clones with random integration or with distorted integration at the target locus show either no left band, no right band or none of both. Plasmids containing cloned sequences of integrated left or right flanking arms including the primer matching sites and gDNAs from parental hPi cells served as controls. (C) Bi-allelic targeted clones hPi-#7, hPi-#8 and hPi-#9 were transfected with hyperactive piggyBac transposase and selected using FIAU. FIAU-resistant, puro-sensitive clones were analyzed by the same PCRs as in (B). (D) Colony formation of bi-allelic targeted clone hPi-#8 at different days of FIAU addition post puromycin withdrawal. At day zero cells were directly transferred from puromycin to FIAU medium. A 1% spike of a HSV thymidine kinase-negative hPi-derived cell line with stable eGFP expression (hPi-GFP) served as control. (E) QRT-PCR analysis of AAT expression levels in undifferentiated hPi iPSC and H9 ESC compared to d17 hepatic differentiated hPi, relative to GAPDH. Data in (E) are represented as mean ± SD with n = 3 biological replicates.
